Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells

Many viruses have evolved strategies of so-called “superinfection exclusion” to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.

characterized in a model of chronic infection, but whether it occurs during the acute phase of JUNV infection remains to be determined.
Here, we show that superinfection exclusion does not occur during acute sequential rounds of infection of either Vero or A549 cells with the Candid1 strain of JUNV. Cells acutely infected by a first round of JUNV infection are still fully permissive for virus internalization, viral RNA synthesis, and translation of viral proteins associated with a second round of JUNV infection harbouring the same Candid1 surface glycoprotein complex (GPC). To the best of our knowledge, these results indicate that JUNV is one of the only viruses that does not exhibit superinfection exclusion by its own kind.

Results and Discussion
We first used a fluorescence microscopy visualization assay to determine whether the JUNV-infected cells allow internalization of new, incoming viral particles (Fig. 1). Entry of fluorescently tagged Junin virus into single cells was assessed using spinning disc confocal fluorescence microscopy according to the experimental design summarized in Fig. 1a. Vero cells were infected at a multiplicity of infection (MOI) of 0.1 and superinfected 16 h later with JUNV particles complexed to an Alexa Fluor 647-labelled non-neutralizing antibody 14,15 to allow visualization of the cell-associated virus particles related to the second round of infection. To discriminate virus particles bound to the cell surface (Fig. 1c, outside) from those that were internalized (Fig. 1c, inside), cells were fixed and incubated without permeabilization with an Alexa Fluor 568-tagged monoclonal antibody specific for the virus glycoprotein complex (GPC) (GB03-A568, outside GPC). After an extensive washing to remove unbound antibodies, cells were fixed and permeabilized, and the nucleoprotein (NP) was detected using an A488-tagged monoclonal antibody. Cells infected during the first round of infection showed extensive and diffuse cytosolic fluorescence NP signal whereas cells infected only during superinfection showed punctae corresponding to bound or internalized particles (Fig. 1b). The relative number of particles associated with superinfected cells was obtained from maximum intensity Z-projections of consecutive optical sections spanning the entire cell volume imaged 500 nm apart and normalized by the area of the cell (Fig. 1d). These results demonstrate that pre-infection of Vero cells did not affect the entry of JUNV particles during superinfection.
The human transferrin receptor (TfR) is considered the main receptor for Junin virus 16 . Here, we investigated whether acute JUNV infection of Vero cells could down-modulate the amount of available TfR. We found that the amount of TfR expressed at the cell surface was not affected by 16 h of infection (Fig. 2b). Consistent with this, the efficiency of receptor-mediated endocytosis of Tf (Fig. 2c) remained stable. Transferrin receptor mRNA levels as well as those of the newly described JUNV entry factor calcium-voltage pump 17 were significantly increased, independently of the MOI used (Fig. 2d,e). Consistent with our results, CD34 + hematopoietic progenitor cells infected with JUNV for 120 h have also been shown to express higher TfR levels 18 .
The Z protein of the Tacaribe virus, another NW arenavirus closely related to JUNV, inhibits viral RNA synthesis by direct interaction with the L viral polymerase 19 . To determine whether superinfecting JUNV might also inhibit its own replication, we used as a marker of infection a recombinant JUNV that expresses enhanced green fluorescent protein (GFP) 20 . JUNV-GFP contains the Candid1 short segment, in which the GPC gene was replaced with GFP, so that its growth in BSR-T7 cells is complemented in trans by expression of Candid1 GPC. The cells producing JUNV-GFP were also infected with a recombinant vaccinia virus encoding T7 polymerase (vTF7-3) because the recombinant JUNV genome is expressed under the control of a T7 promoter 21 . Vero cells were first infected with JUNV at a MOI of 5 for 16 h and then superinfected with JUNV-GFP for an additional 8 h. Cells were then lysed and RNA extracted for reverse-transcription real-time quantitative PCR (RT-qPCR; see experimental design in Fig. 3a). Because the recombinant JUNV-GFP virus does not contain the sequence coding for GPC, GPC RNA detection by RT-qPCR was used to monitor the first round of infection with JUNV (Fig. 3b). RT-qPCR specific for the GFP sequence was used to evaluate the efficiency of replication in the second round of infection with JUNV-GFP (Fig. 3c). The results showed that a similar amount of GFP RNA from JUNV-GFP was generated in the second round regardless of a first round of infection with JUNV.
Because Vero cells are deficient for interferon α and β production, we also tested superinfection in the interferon-competent A549 cell line (Fig. 3d,e) and confirmed that GFP RNA expression from the second infection was identical whether or not the cells were previously infected with JUNV, further suggesting an absence of superinfection exclusion as measured by the amount of replication during acute JUNV infection.
Because the Z protein of the OW arenavirus LCMV has also been suggested to inhibit translation 22 , we took advantage of the JUNV/JUNV-GFP superinfection system (Fig. 4a) to investigate effects during superinfection on viral translation at the single cell level (Fig. 4). Flow cytometry allowed discrimination of Vero cells infected during the first round (GPC antibody staining; Fig. 4b) and the second round (GFP protein expression; Fig. 4c). Prior JUNV infection had no effect on the number of GFP-expressing cells, even when taking into account only the highest GPC-expressing cells from the first infection ( Fig. 4d) or using higher MOI (Fig. 4e). Moreover, cells infected during the second round (GFP+ cells) exhibited similar GFP fluorescence intensity regardless of whether they were infected (GPC-A647 positive) or not (GPC-A647 negative) during the first round of infection (Fig. 4f). These results suggest that JUNV is fully capable of superinfection.
Overall, our study shows that acutely infected cells remain permissive to a second round of JUNV infection to the same extent as non-infected cells. Our JUNV/JUNV-GFP detection system allowed us to monitor acute infection and readily discriminate between the first and second rounds of infection. We note that it was not feasible to determine with this assay whether the newly formed JUNV-GFP particles were infectious because they lacked GPC, which was replaced by GFP; thus, they were unable to bind to the natural receptor in the host cell.
Previous studies have shown that JUNV can induce superinfection exclusion, but these experiments by Ellenberg et al. were performed in cells chronically infected by JUNV for several years 10,11 . In one of these reports 10 , the authors suggested that failure to superinfect chronically infected Vero cells was related to the presence of NP and proposed that inhibition could occur at a step between replication and translation 10 . In the second study 11 , Ellenberg et al. explained superinfection exclusion by the fact that chronically infected BHK-21 cells diminished synthesis of superinfecting virus proteins, along with an inhibition of JUNV budding mediated by the overexpression of Tsg101 11 . In contrast, our superinfection experiments were exclusively conducted during acute infection of Vero or A549 cells. We showed that viral entry Data are average ± SD from two independent experiments in which more than 200 particles were counted. and viral genome replication and protein synthesis were normal or even slightly higher during the second round of infection. One possible explanation for the differences between our findings with acutely infected cells and those that Ellenberg et al. obtained for chronically infected Vero cells 12 is that the cells they used do not express GPC or produce infectious particles and are resistant to the cytopathic effects of Junin virus, suggesting that failure to superinfect might be related to a host perturbation. Nevertheless, their results in BHK-21 chronically infected cells 13 are consistent with our own observation with acutely infected Vero cells, showing that viral transcription and translation were not perturbed during superinfection. Although particle assembly and release was diminished in the chronically infected BHK-21 cells, it remains to be determined whether a similar perturbation is manifested in the acutely infected Vero cells.
In conclusion, our results highlight an important mechanistic difference in superinfection exclusion, not only between the Junin virus and other closely related OW arenaviruses 11 but also with the large number of other virus family members that show superinfection exclusion.

Virus production and labelling. Junin virus or JUNV refers to the non-pathogenic vaccine strain
Candid1 (obtained from the Bavari laboratory at the US Army Medical Research Institute of Infectious Diseases). Wild-type JUNV was produced and labelled as previously described 15 . Briefly, JUNV stock was incubated with the non-neutralizing mouse monoclonal antibody LD05 raised against the JUNV envelope glycoprotein (4 μ g ml −1 ) and coupled to Alexa Fluor 647 (Life Technologies) for 30 min at 25 °C. The virus-dye mixture was then gently applied on top of a 10% Optiprep (Sigma-Aldrich) cushion and ultracentrifuged at 150,000 × g for 2 h. The pellet containing the JUNV-A647-labelled particles was resuspended in virus media. The JUNV-GFP virus was produced as follows: BSR-T7 cells were infected with the vTF7-3 vaccinia virus 21 for 1 h and then simultaneously transfected with the plasmids pJCd1L, pJCd1S-DGPC:GFP, and pC-GPC as described in 20 , using TransIT-2020 Transfection Reagent (Mirus). After 5 h, cells were washed and re-incubated in virus media.
Immunofluorescence and light microscopy. Vero cells grown on coverslips, infected or non-infected, were fixed with 4% paraformaldehyde and incubated (without permeabilization) with the GB03-A568 antibody specifically recognizing the glycoprotein GPC of JUNV. Then, cells were fixed, permeabilized with 0.5% bovine serum albumin and 0.05% saponin in phosphate-buffered saline, and stained with the mouse monoclonal anti-NP antibody SA02-A647 and 4' ,6-diamidino-2-phenylindole (DAPI). The samples were mounted on glass slides and imaged on a Zeiss AxioObserver.Z1 inverted microscope mounted with a spinning disc head (Yokogawa), a QuantEM:512SC EMCCD camera (Photometrics), and a 63 × 1.4 NA oil objective (Zeiss). Each acquisition corresponded to stacks spaced by 0.5 μ m that spanned the whole cell volume, and images were analysed using ImageJ (version 1.48d). RNA analysis. Total RNA from cells was purified using the RNeasy Mini kit (Qiagen). RNA was then reverse-transcribed into complementary DNA using the SuperScript VILO cDNA Synthesis Kit (Life Technologies). RT-qPCR amplification was performed using FastStart Universal SYBR Green Master (Rox) (Roche). Complementary DNA of viral GPC, GFP, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected using the following specific forward and reverse primers (5′→ 3′), respectively: viral GPC, cctagcgcttgcaggaagatcc and caccagctcatatctgctggatg; GFP, aagctgaccctgaagttcatctgc and cttgtagttgccgtcgtccttgaa; human transferrin receptor protein 1 (TFRC) caagctagatcagcattctctaacttg and PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems), and amplification cycles were set as follows: PCR initial activation step, 10 min at 95 °C; 40 cycles of denaturation and combined annealing/extension, 15 s at 95 °C; and 1 min at 60 °C. Fluorescence data collection was achieved at the end of each cycle, and a melting curve showing primer specificity was included at the end of each reaction.
Transferrin uptake. Transferrin uptake assay was performed as in 23 . Briefly, Vero cells were pre-incubated at 4 °C or 37 °C for 15 min followed by incubation for 10 min and at the same temperatures with 5 μ g ml −1 Transferrin-Alexa Fluor 647 (Tf-A647, Life Technologies). After incubation, the 37 °C samples were cooled on ice and rinsed with cold PBS, and 4 °C and 37 °C samples were briefly incubated twice with 150 mM NaCl, 1 mM MgCl 2 , 0.125 mM CaCl 2 , 0.1 M glycine pH 2.5 to remove the surface bound Tf-A647. A sample incubated at 4 °C with Tf-A647 but not treated with the acid wash was used to estimate the amount of fluorescent transferrin bound at the cell surface. Cells were then resuspended in 200 μ l of PBS containing 1% bovine serum albumin and 0.5 mM EDTA at 4 °C. Measurement of the fluorescence intensity, reflecting the extent of Tf endocytosis for each cell, was determined by flow cytometry using the 633 nm laser line of the FASCSCanto II (BD Biosciences).
Virus infectivity assays. JUNV-infected cells were identified by flow cytometry. For flow cytometry, cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.5% bovine serum albumin and 0.05% saponin in phosphate-buffered saline, followed by incubation with a GD01 mouse monoclonal antibody (2 μ g ml −1 ) specific for the JUNV glycoprotein and coupled to Alexa Fluor 647. Acquisition of fluorescence intensity of the infected cells was performed on a FACSCanto II (BD Biosciences) using 488 nm (for GFP detection) and 640 nm (for Alexa Fluor 647 detection) lasers. Analysis of the percentage of infected cells was completed using FlowJo (Treestar Inc).